UHRF1Gene Targeted Editing In Human Embryonic Kidney Epithelial Cells Based On CRISPR/Cas9System

CRISPR（clustered regularly interspaced short palindromic repeats）system is an adaptiveimmunity mechinary ubiquitously in bacteria and archaea, aiming to defend genomeintegrity by recognizing and silencing bacteriophage DNA. CRISPR system consists ofrepeat sequences (CRISPR array) and upstream CRISPR associated protein family (Cas)operon. Exogenous sequences are recognized and inserted into the CRISPR array betweenrepeat sequences termed proto-spacers, thus CRISPR system acquire the ability todistinguish between endogenous and exogenous sequences. CRISPR system is classifiedinto three categories on the basis of their different components: type I, type II and type III.As the newest genome editing technology, CRISPR/Cas9system is developed based on thetype II system because of its minimum components. Compared with the relatively maturegenome editing technology, ZFN (Zinc-finger nuclease) and TALEN (transcriptionactivator-like effector nucleases), CRISPR/Cas9system is easier to engineer, more scalableand affordable. Human UHRF1(ubiquitin-like with PHD and RING finger domains1)protein is a multifunctional domains protein expressed by UHRF1gene. It participates inmany biological processes including gene regulation, methylation inheritance, cell cycleprogression and even cancer development, but its detailed mechanisms are notentirely clear. Construction of UHRF1gene knockout human cell model will help for morein-depth study of UHRF1protein function.OBJECTIVE: Establish UHRF1knockout HEK293T cell strains through CRISPR/Cas9system with low off-target tendency and preliminarily analysis its effects on HEK293T.METHODS:①Design gRNA targeting to a exon of UHRF1and cloning into gRNAvector. Co-transfection the constructed gRNA vector and hCas9vector into humanHEK293T cells. Screening the cells with puromycin resistance and establishmentmonoclonal cell strains;②Identification the UHRF1knockout cell strains by western blotand targeted sites DNA sequencing;③Verification of the global DNA methylationinduced by UHRF1knockout through LUMA assay;④Detection of the influence on cellcycle and cell apoptosis by flow cytometry on HEK293T knockout cells.RESULTS: Obtained specific target on UHRF1gene and construct gRNA vectorsuccessfully. Co-transfected it with hCas9vector and established34monoclonal cellstrains. Western blot identified three monoclonal cell strains with significantly declinedUHRF1protein expression level. Sequencing results showed that both of them are heterozygous at UHRF1gene locus. Chosen a HEK293T cell strain with the lowestUHRF1expression and confirmed its genome-wide hypomethylation by LUMA assay.Overexpress the UHRF1in UHRF1gene knockout HEK293T cell stain could rescue thegenome methylation level of the strain. Flow cytometry showed that declined UHRF1protein expression in HEK293T cells exhibited prolonged G1-phase with a high apoptosispopulation.CONCLUSIONS: UHRF1gene specific knockout monoclonal HEK293T cell strainswere first established by CRISPR/Cas9system which displays a genome-widehypomethylation. Our results provide an effective cell model for further studying ofUHRF1epigenetic regulation with potential value on tumor diagnosis and drug discovery.